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Blood, Vol. 112, Issue 10, 3949-3958, November 15, 2008
IL-3 induces a Pim1-dependent antiapoptotic pathway in primary human basophils
Blood Didichenko et al.
112: 3949
Supplemental materials for: Didichenko et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 97 KB)
- Figure S1. Differences in the strength and the duration of activation of signaling molecules following stimulation of basophils with IL-3 and IL-5 (JPG, 41.3 KB)
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Freshly isolated basophils were stimulated in serum-free medium with IL-3 or IL-5 at saturating concentrations (50 ng/ml) for the time periods indicated and analyzed as described in Document 1 above. Activation of Stat3/5 and PKB was examined by immunoblotting with phospho-specific antibodies. Both cytokines induce a comparable initial activation of Stat5 and PKB. However, IL-3-mediated activation of Stat5 is more prolonged. The strength and the duration of Stat3 activation by IL-3 is much higher than that induced by IL-5.
- Figure S2. The degree of Pim1 protein expression reflects the level of caspase 3 inhibition in human neutrophils and eosinophils upon stimulation with cytokines of the GM-CSF family (JPG, 28.9 KB)
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Freshly isolated neutrophils and eosinophils were cultured in medium alone or in the presence of the cytokine indicated (all at 50 ng/ml) during 16 h. Protein levels of Pim 1, procaspase 3 and activated caspase 3 were analyzed by immunoblotting.
- Figure S3. Inhibitors of PI3-kinase suppress the IL-3-induced transient activation of PKB in a dose-dependent manner (JPG, 41.6 KB)
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Freshly isolated basophils were preincubated with wortmannin (WT) or LY294002 (LY) at the concentrations indicated in the figure for 30 min before stimulation with IL-3 (50ng/ml; stimulation time 5 min). Cells stimulated with buffer or IL-3 without inhibitors were used as controls. Protein samples were prepared as described in Document 1 above. Activation of Stat3/5 and PKB was analyzed by immunoblotting using phospho-specific antibodies. PI3-kinase inhibitors do not influence IL-3-induced Stat3/5-activation. Activation of PKB is significantly inhibited by 10 nM WT, and completely abolished by 100 nM WT. LY294002 is effective at 30 µM, whereas at 10 µM only a decrease in PKB activation is observed. Lower concentrations of WT (1 nM) or LY (1 µM) have no inhibitory effects on PKB activation by IL-3.
- Figure S4. Retarded electrophoretic mobility of TAT-Pim1(wt) is due to autophosphorylation (JPG, 35 KB)
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Purified TAT-Pim1 fusion proteins were treated or not with λ-protein phosphatase for 30 min or 1h, as described in Document 1 above. Proteins were separated by PAGE and visualized on a gel by Coomassie G 250 stain. λ-PPase treatment increases the electrophoretic mobility of TAT-Pim1(wt) which becomes similar to that of TAT-Pim1(KD). These results are consistent with the previous report by Bullock et al. (JBC, 280:41675-41682, 2005) demonstrating that Pim1 undergoes autophosphorylation upon expression in E. coli.
- Figure S5. Dose-dependent inhibition of caspase 3 activation by TAT-Pim1(wt) but not TAT-Pim1(KD) (JPG, 34.7 KB)
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Basophils cultured in medium without IL-3 were transduced with TAT-Pim1 fusion proteins at the indicated concentrations. Fusion proteins were added to the cells every 6 h. At 24h posttransduction, cellular proteins were analyzed by immunoblotting using anti-HA antibody and anti-cleaved caspase 3 antibody. Basophils cultured in medium alone or with IL-3 without fusion proteins are included as controls. A concentration of 20 µg/ml TAT-Pim1(wt) was effective in inhibiting caspase 3 activation, while lower concentrations had only a minimal or no effect. No significant changes in caspase 3 activation were observed in cells transduced with TAT-Pim1(KD) compared to control cells.
- Figure S6. Intracellular localization of TAT-Pim1 fusion proteins in basophils (JPG, 48.8 KB)
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Basophils were transduced with TAT-Pim1 fusion proteins (20 µg/ml) for 24h. Indirect immunofluorescence was carried out using double staining using anti-HA antibody (12C5) and anti-cleaved caspase antibody (Asp175)(5A1) as described in Document 1 above. DNA was stained with DAPI. The majority of basophils were transduced with fusion proteins. Cells positive for active cleaved caspase 3 are marked with arrowheads. The distribution of TAT-Pim1(wt) as well as TAT-Pim1(KD) within the cells was uniform with punctuate inclusions in the cytoplasm.
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